We propose to investigate the interactions with DNA of two classes of protein which are required for DNA replication: the helicases and helix destabilizing proteins. In particular we will study the rep protein (a helicase) coded for by the bacterium E. coli, and two helix destabilizing proteins, the E. coli single stranded binding protein (SSB) and the bacteriophage coded T4 gene 32 protein. The rep protein, with the aid of the SSB protein, catalyzes the ATP dependent unidirectional unwinding of double stranded DNA, a process which requires translocation of rep along the DNA and is fundamental to DNA replication. Physical-biochemical techniques will be used to quantitatively investigate the equilibrium and kinetic binding properties of the purified proteins to DNA as a function of solution variables. The methods to be used to study the interactions include sedimentation, chromatography, gel electrophoresis, spectroscopy and stopped-flow kinetics. The mechanism of DNA unwindig, catalyzed by rep, will also be investigated, focusing on the rates of unwinding and the processive action of rep. The unwinding reaction can be studied in the absence of DNA synthesis and hance provides an excellent system for physical-biochemical studies. These studies are specifically directed to understand the rep catalyzed DNA unwinding reaction and the mechanism of the ATP driven rep translocation, however they will also reveal thermodynamic details which will increase our general knowledge of the factors which stabilize protein-nucleic acid interactions. Furthermore, the mechanistic information obtained form studies of the kinetics of these protein-DNA interacations should be useful in studies of other proteins which must also translocate along DNA (driven thermally or by hydrolysis of ATP). With this research we begin our attempt to understand complex multiprotein-DNA systems (such as those that occur in DNA replication and recombination) at the level of their particular protein-protein and protein-DNA interactions, with the goal of understanding the basis of their control. Since DNA replication is fundamental to cell growth, and understanding of its details will undoubtedly increase our chances for determining how it malfunctions in diseases such as cancer.